The goals of Project 2 are to develop a detailed picture of human disease-associated RecQ helicase protein function, and to use this information to delineate the role of these RecQ helicases in human tumor cell proliferation, survival and the response to chemotherapy. In research to date we have focused on functional analyses of the human Werner syndrome protein WRN. In the proposed research we extend these analyses to encompass all three disease-associated human RecQ helicases, the WRN, BLM and RECQL4 proteins associated with the heritable deficiency/genetic instability/cancer predisposition syndromes Werner syndrome (WRN), Bloom syndrome (BLM) and Rothmund-Thomson (RTS) syndrome, respectively. Aim 1. Develop a comprehensive proteomic description of the human disease-associated WRN, BlM and RECQl4 human RecQ helicases. We will identify functionally important RecQ helicase protein associations and post-translational modifications (PTM's) by using quantitative affinity purification mass spectrometry, shRNA-mediated epistatic interaction mapping and functional analyses of post-translational modification (PTM) site mutants. Aim 2. Determine functional requirement for WRN, BlM and RECQL4 in DNA metabolism. We will determine the role of each of these RecQ helicases in DNA replication, translesion DNA synthesis (TLS), homology-dependent recombination (HDR) and DNA break repair using high resolution assays. Aim 3. Determine functional redundancy among WRN, BlM and RECQL4 in DNA metabolism, and clinically relevant synthetic interactions with additional genes/proteins, drugs or small molecules. We will identify unique and redundant functions of the 'disease-associated human RecQ helicases, and synthetic interactions between disease-associated human RecQ helicases, their interacting proteins, and chemotherapeutic agents, drugs or small molecules that can beused to selectively kill RecQ-deficient human tumors.